生物合成聚3-羟基丙酸酯的研究进展

聚3-羟基丙酸酯(P3HP)作为聚羟基脂肪酸酯家族(PHAs)中的新型热塑性塑料,具有生物降解性和生物相容性等优点。目前,未见野生微生物可以合成P3HP的报道,生产途径主要为化学法和生物法。其中,通过化学法或添加3-HP单体及其结构类似物作为前体的P3HP合成效率低、成本高且不具环保性;而通过构建和改造工程菌的生物代谢途径,能够利用廉价、可再生的碳源,已经逐渐成为研究热点。文中综述了国内外P3HP生物合成研究进展,并对甘油途径、丙二酸单酰辅酶A(Malonyl-Co A)途径和β-丙氨酸途径等合成方法进行了优缺点分析,为生物合成P3HP的深入研究奠定理论基础。     

应用聚合酶链式反应快速特异鉴定假单胞菌和伯克霍尔德氏菌(R)-3-羟基酯酰载酯蛋白-辅酶A转酰基酶基因

聚羟基脂肪酸酯(PHA)是一类具有广泛应用前景的可降解生物塑料。因其可以以葡萄糖等廉价底物直接发酵生产PHA而日益受到重视。目前的研究表明在积累中长链PHA的假单胞菌中,由phaG基因编码的(R)-3-羟基酯酰载酯蛋白-辅酶A转酰基酶(PhaG)起关键作用,但目前为止对该蛋白还知之甚少。通过聚合酶链式反应(PCR)建立了一种快速、特异鉴定phaG基因的方法,应用该方法成功地从两株积累不同PHA的假单胞菌Pseudomonas stutzeri 1317和Pseudamanas nitroreducens 0802中分别克隆得到phaG基因,并在phaG基因突变株Pseudomonas putida PHAGx-21中表达成功。同时,还首次报道了从非假单胞菌菌株Burkholderia caryophylli AS 1．2741中鉴定得到phaG基因,提示PhaG介导的中长链PHA合成途径作为一种通用的代谢模式在细菌中广泛存在,为进一步实现从廉价的非相关底物合成中长链PHA提供了必要的分子生物学基础。     

重组大肠杆菌合成聚(3-巯基丙酸)和聚(3-羟基丁酸-3-巯基丙酸)的研究

利用Clostridium acetobutylicum的丁酸激酶基因 (buk) 和磷酸转丁酰基酶基因(ptb),以及Thiocapsa pfennigii的PHA合成酶基因,设计了一条能够合成多种聚羟基烷酸的代谢途径,用构建的质粒转化大肠杆菌,获得了重组大肠杆菌菌株.前期的研究表明,在合适的前体物条件下,该重组大肠杆菌能够合成包括聚羟基丁酸、聚(羟基丁酸-戊酸)等多种生物聚酯[Liu and Steinbüchel, Appl. Environ. Microbiol. 66739-743].利用该重组大肠杆菌,通过生物催化作用合成了3-巯基丙酸的同型共聚酯,同时利用该重组大肠杆菌还获得了含3-巯基丙酸单体的多种异型共聚物.实验首先研究了3-巯基丙酸对大肠杆菌生长的影响,在此基础上优化了培养过程中添加3-巯基丙酸的时机和浓度,结果表明,在实验的条件下,细胞合成聚(3-巯基丙酸)可达6.7%(占细胞干重),合成聚(3-羟基丁酸-3-巯基丙酸)(分子中3-巯基丙酸3-羟基丁酸=31)可达24.3%.实验进一步研究了同时或分别表达以上3个基因的重组大肠杆菌合成聚合物的能力,结果表明只有当3个基因同时表达时才能合成聚合物,说明3个基因对合成过程是必须的,从而表明了合成途径是按照设计的路线进行的.还通过GC/MS、GPC、IR等手段对合成的化合物进行了定性的研究.聚(3-巯基丙酸)或聚(3-羟基丁酸-3-巯基丙酸)等聚酯属于一类新型生物聚合物,它在分子骨架中含有硫酯键,不同于聚羟基烷酸酯的氧酯键,从而具有显著不同的物理、化学、光学等性质和具有重要的潜在应用价值.     

重组大肠杆菌合成聚（3-巯基丙酸）和聚（3-羟基丁酸-3-巯基丙酸）的研究

利用Clostridium acetobutylicum的丁酸激酶基因 (buk) 和磷酸转丁酰基酶基因(ptb),以及Thiocapsa pfennigii的PHA合成酶基因,设计了一条能够合成多种聚羟基烷酸的代谢途径,用构建的质粒转化大肠杆菌,获得了重组大肠杆菌菌株。前期的研究表明,在合适的前体物条件下,该重组大肠杆菌能够合成包括聚羟基丁酸、聚（羟基丁酸戊酸）等多种生物聚酯［Liu and Steinbüchel, Appl. Environ. Microbiol. 66:739743］。利用该重组大肠杆菌,通过生物催化作用合成了3巯基丙酸的同型共聚酯,同时利用该重组大肠杆菌还获得了含3-巯基丙酸单体的多种异型共聚物。实验首先研究了3巯基丙酸对大肠杆菌生长的影响,在此基础上优化了培养过程中添加3-巯基丙酸的时机和浓度,结果表明,在实验的条件下,细胞合成聚（3-巯基丙酸）可达6.7%(占细胞干重),合成聚（3-羟基丁酸—3-巯基丙酸）（分子中3-巯基丙酸:3-羟基丁酸=3:1）可达24.3%。实验进一步研究了同时或分别表达以上3个基因的重组大肠杆菌合成聚合物的能力,结果表明只有当3个基因同时表达时才能合成聚合物,说明3个基因对合成过程是必须的,从而表明了合成途径是按照设计的路线进行的。还通过GC/MS、GPC、IR等手段对合成的化合物进行了定性的研究。聚（3-巯基丙酸）或聚（3-羟基丁酸-3-巯基丙酸）等聚酯属于一类新型生物聚合物,它在分子骨架中含有硫酯键,不同于聚羟基烷酸酯的氧酯键,从而具有显著不同的物理、化学、光学等性质和具有重要的潜在应用价值。     

聚3-羟基丁酸酯（PHB）生物降解过程的研究

利用DS9701菌株对聚3-羟基丁酸酯(PHB)膜进行降解,对降解到不同程度的PHB膜采用扫描电子显微镜观察其表面形态结构的变化,并对其降解产物进行分析测定.结果表明,PHB的生物降解首先发生在PHB表面的非晶部分,随后结晶部分开始降解,并且降解首先发生在球晶的中心部分.DS9701菌株所产生的PHB解聚酶主要降解PHB的第二个酯键,降解产物为二聚体.     

植物3-羟基-3-甲基戊二酰辅酶A还原酶基因研究进展

细胞分裂素、赤霉素、脱落酸、叶绿素、萜类等类异戊二烯物质,是植物中广泛存在的一类代谢产物,在植物生长发育过程中起着非常重要的作用。一些萜类化合物作为药物的合成前体或有效的药用成分在工农业及医药生产上具有重要的经济价值。类异戊二烯物质主要通过甲羟戊酸代谢途径中的一系列酶催化合成,其中,3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR)是该代谢途径中的第一个关键限速酶,能够将3-羟基-3-甲基戊二酰辅酶A转化成中间代谢产物甲羟戊酸。对植物HMGR基因的克隆、酶结构和功能分析、基因组织表达及调控等方面进行了综述,旨在为其在重要农作物的遗传改良、代谢产物工程植物创制以及植物亲缘关系分析中的应用等研究提供理论依据。     

酿酒酵母gpd1和hor2基因在大肠杆菌中的共表达

利用途径工程的方法,在大肠杆菌中构建一条新的产甘油的代谢途径。从酿酒酵母(Saccharomyces cerevisiae)克隆3-磷酸甘油脱氢酶基因(gpd1)和3-磷酸甘油酯酶基因(hor2),并将两个基因串连到启动子trc的下游,构建由trc启动子控制的能高效表达的多顺反子重组质粒pSE-gpd1-hor2,将重组质粒导入大肠杆菌BL21菌株中,构建得到的重组菌株GxB-gh能将葡萄糖转化为甘油。结果表明重组菌株GxB-gh以葡萄糖为底物进行发酵,甘油产量为46．67g／L,葡萄糖的转化率为42.87％。这为利用工程菌绿色生产甘油进行了前期的探索,也为进一步构建能生产1,3-丙二醇的工程菌打下了良好的基础。     

应用聚合酶链式反应快速特异鉴定假单胞菌和伯克霍尔德氏菌（R）-3-羟基酯酰载酯蛋白-辅酶A转酰基酶基因

聚羟基脂肪酸酯 (PHA)是一类具有广泛应用前景的可降解生物塑料。因其可以以葡萄糖等廉价底物直接发酵生产PHA而日益受到重视。目前的研究表明在积累中长链PHA的假单胞菌中 ,由phaG基因编码的（R）3 羟基酯酰载酯蛋白 辅酶A转酰基酶 (PhaG)起关键作用 ,但目前为止对该蛋白还知之甚少。通过聚合酶链式反应 (PCR)建立了一种快速、特异鉴定phaG基因的方法 ,应用该方法成功地从两株积累不同PHA的假单胞菌Pseudomonasstutzeri 1317和Pseudomonasnitroreducens 080 2中分别克隆得到phaG基因 ,并在phaG基因突变株PseudomonasputidaPHAG_N-21中表达成功。同时 ,还首次报道了从非假单胞菌菌株Burkholderiacaryophylli AS 1.274 1中鉴定得到phaG基因 ,提示PhaG介导的中长链PHA合成途径作为一种通用的代谢模式在细菌中广泛存在 ,为进一步实现从廉价的非相关底物合成中长链PHA提供了必要的分子生物学基础。     

hsa-miR-342-3p生物信息学分析

通过生物信息学方法预测hsa-miR-342-3p靶基因及其功能机制。检索Pub Med有关hsa-miR-342-3p的研究报道并进行功能分析;检索miRBase获取hsa-miR-342-3p序列;通过Target Scan,Pictar和PITA数据库预测靶基因并取交集,对其进行组织和疾病特异性表达谱分析、功能富集分析(GO enrichment analysis)、信号转导通路富集分析(Pathway enrichment analysis)和蛋白质相互作用网络分析(PPI analysis)。结果发现:hsa-miR-342-3p序列在多物种间具有高度保守性;hsa-miR-342-3p在肾脏组织和急性淋巴细胞性白血病、乳腺癌疾病中表达水平较高(RPM≥1 000);预测得到14个hsa-miR-342-3p靶基因;靶基因分子功能分别富集于转化生长因子活性、DNA结合和蛋白激酶激活等(P0.05);hsa-miR-342-3p靶基因GO生物学过程主要集中于大分子代谢抑制,肺部组织发育、呼吸系统发育及管状组织发育建成(P0.05);细胞信号通路主要富集于TGF-Beta信号通路、细胞因子、受体作用信号通路及前列腺疾病信号通路(P0.01)。hsa-miR-342-3p在体内分布广泛,预测的靶向TGF-Beta信号通路可能在疾病发生中发挥重要调控作用,是具有潜在研究价值的生物学靶标。     

黄杆菌菌株RC2-3基因组生物信息学分析及高效降解岩藻多糖功能基因的挖掘

以从海带中筛选获得的一株具有降解岩藻多糖能力的黄杆菌菌株RC2-3为研究对象，该菌株产的岩藻多糖酶可以高效降解不同来源的岩藻多糖。为进一步探究菌株RC2-3降解岩藻多糖的机制，推动岩藻寡糖的酶法生产，采用Illumina测序技术对菌株RC2-3进行基因组测序、基因功能注释和碳水化合物活性酶注释以及岩藻多糖降解相关基因的生物信息学分析。结果表明，黄杆菌菌株RC2-3基因组全长3 414 532 bp,共编码2 967个基因，GC含量为30.92%。经碳水化合物活性酶数据库注释获得213个基因，与岩藻多糖降解有关的包括7个岩藻糖结合结构域的基因；2个β-D-岩藻糖苷酶(EC 3.2.1.38)基因；2个属于GH141家族的α-L-岩藻糖苷酶(EC 3.2.1.51)基因；12个属于GH29家族的α-1, 3/1, 4-L-岩藻糖苷酶(EC 3.2.1.111)基因；9个属于GH95家族的α-1, 2-L-岩藻糖苷酶(EC 3.2.1.63)基因。此外，通过与已报道的蛋白序列比对发现，岩藻多糖酶基因RC2.3＿GM001247编码的蛋白序列与FunA蛋白序列同源性达到70.98%,岩藻多糖酶...     

Purification and Characterization of Fungal Poly(3-hydroxybutyrate) Depolymerase from Paecilomyces lilacinus F4-5 and Enzymatic Degradation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Film

Summary Poly(3-hydroxybutyrate) [P(3HB)] depolymerase was purified from a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]-degrading fungus, Paecilomyces lilacinus F4-5 by hydrophobic and ion exchange column chromatography, and showed a molecular mass of 45 kDa. The optimum temperature and pH of the P(3HB) depolymerase were 50 °C and 7.0, respectively. The enzyme was stable for at least 30 min at temperatures below 40 °C, while the activity abruptly decreased over 55 °C. Enzymatic P(3HB-co-3HV) degradation showed a similar degradation pattern to that of film overlaid by fungal hyphae. It reflects that the fungal degradation of P(3HB-co-3HV) in soil is mainly caused by extracellular depolymerases.     

烟芽夜蛾囊泡病毒3h株编码的3H-117蛋白能干扰AcMNPV的口服活性

[目的]囊泡病毒是一类体液传播的昆虫病毒,具有特殊的致病历程和良好的生防应用潜力.烟芽夜蛾囊泡病毒3h株(Heliothis virescens ascovirus 3h,HvAV-3h)是我国分离的第一株囊泡病毒,其编码的3H-117蛋白被报道为HvAV-3h病毒粒子的结构蛋白.[方法]为了进一步探究3h-117基因...     

Biosynthesis and Characterization of Poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) from Palm Oil Products in a Wautersia eutropha Mutant

Palm kernel oil, palm olein, crude palm oil and palm acid oil were used for the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by a mutant strain of Wautersia eutropha (formerly Ralstonia eutropha) harboring the Aeromonas caviae polyhydroxyalkanoate (PHA) synthase gene. Palm kernel oil was an excellent carbon source for the production of cell biomass and P(3HB-co-3HHx). About 87% (w/w) of the cell dry weight as P(3HB-co-3HHx) was obtained using 5 g palm kernel oil/l. Gravimetric and microscopic analyses further confirmed the high PHA content in the recombinant cells. The molar fraction of 3HHx remained constant at 5 mol % regardless of the type and concentration of palm oil products used. The small amount of 3HHx units was confirmed by ¹³C NMR analysis. The number average molecular weight (M_n) of the PHA copolymer produced from the various palm oil products ranged from 27 0000 to 46 0000 Da. The polydispersity was in the range of 2.6–3.9.     

Presence of indole-3-acetic acid in chloroplasts ofNicotiana tabacum andPinus sylvestris

The compartmentation and metabolism of indole-3-acetic acid (IAA) was examined in protoplasts derived from needles ofPinus sylvestris L., leaves of normal plants ofNicotiana tabacum L., leaves ofN. tabacum plants carrying the T-DNA gene 1 (rG1 plants) and leaves ofN. tabacum plants carrying the T-DNA gene 2 (rG2 plants) by using a rapid cell-fractionation method. In all tissues, 30%–40% of the IAA pool was located in the chloroplast, while the remainder was found in the cytosol. Quantitative analysis of indole-3-ethanol (IEt) showed that in bothPinus andNicotiana the IEt pool was located exclusively in the cytosol. The only plant that contained endogenous indoleacetamide (IAAm) was therG1-mutant ofN. tabacum, expressing theAgrobacterium tumefaciens T-DNA gene 1. Cellular fractionation of protoplasts from this transgenic plant showed that the entire IAAm pool was located in the cytosol. Feeding experiments utilizing [5-³H]tryptophan, [5-³H]IEt, [1′-¹⁴C] and [2′-¹⁴C]IAA demonstrated that the biosynthesis and catabolism of IAA occurred in the cytosol in bothPinus and in the wild type and the different mutants ofNicotiana. Furthermore, the biosynthesis of IAAm in therG1 plants was also shown to be localized in the cytosol.     

Endogenous indoles and the biosynthesis and metabolism of indole-3-acetic acid in cultures of Rhizobium phaseoli

Gas chromatography-mass spectrometric analyses of purified extracts from cultures of Rhizobium phaseoli wild-type strain 8002, grown in a non-tryptophan-supplemented liquid medium, demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol (IEt), indole-3-aldehyde and indole-3-methanol (IM). In metabolism studies with ³H-, ¹⁴C- and ²H-labelled substrates the bacterium was shown to convert tryptophan to IEt, IAA and IM; IEt to IAA and IM; and IAA to IM. Indole-3-acetamide (IAAm) could not be detected as either an endogenous constituent or a metabolite of [³H]tryptophan nor did cultures convert [¹⁴C]IAAm to IAA. Biosynthesis of IAA in R. phaseoli, thus, involves a different pathway from that operating in Pseudomonas savastanio and Agrobacterium tumefaciens-induced crown-gall tumours.Abbreviations IAA indole-3-acetic acid - IAld indole-3-aldehyde - IAAm indole-3-acetamide - IEt indole-3-ethanol - IM indole-3-methanol - HPLC-RC high-performance liquid chromatography-radio counting - GC-MS gas chromatography-mass spectrometry     

Production of poly-D(-)-3-hydroxybutyrate and poly-D(-)-3-hydroxyvalerate by strains ofAlcaligenes latus

Alcaligenes latus strains can accumulate poly-D(-)-3-hydroxybutyrate (PHB) up to about 85% of cell dry weight. The abilities to store poly-D(-)-3-hydroxyvalerate (PHV) of three strains ofA. latus were investigated. With Na-propionate as PHV precursor, strainA. latusDSM 1122 had better PHV accumulation ability than strainsA. latusDSM 1123 and 1124. StrainA. latus DSM 1123 could store PHV when Na-valerate but not Na-propionate served as the PHV precursor. PHB and PHV accumulation byA. latus DSM 1124 rapidly increased when propionic acid and acetic acid were together added to the fermentor. This increase was not obtained in the culture shaker flask and fermentor growing the same strain when Na-propionate alone served as a PHV precursor.     

Purification and characterization of the NADH-dependent (S)-specific 3-oxobutyryl-CoA reductase from Clostridium tyrobutyricum

An NADH-dependent (S)-specific 3-oxobutyryl-CoA reductase from Clostridium tyrobutyricum was purified 15-fold with a yield of 46%. It was homogeneous by gel electrophoresis after three chromatographic steps. The apparent molecular mass was estimated by column chromatography to be 240 kDa. SDS-gel electrophoresis revealed the presence of 33 kDa subunits. Substrates of the enzyme were ethyl and methyl 3-oxobutyrate, 3-oxobutyryl-N-acetylcysteamine thioester, and 3-oxobutyryl coenzyme A. The specific activities were 340 and 10 U (mg protein)^-1 for the reduction of 3-oxobutyryl coenzyme A and ethyl 3-oxobutyrate, respectively; the Michaelis constants were 300 M and 300 mM, respectively. The identity of 12 N-terminal amino acid residues was determined. The ezmyme was used in a preparative reduction of substrate, yielding ethyl (S)-3-hydroxybutyrate (>99% enantiomeric excess).     

Fed-batch culture ofAeromonas hydrophila for the production of poly(3-hydroxubutyrate-co-3-hydroxyhexanoate) using two carbon sources

To produce polyhydroxyalkanoate (PHA) copolymer which consists of 3-hydroxy-butyrate (3HB) and 3-hydroxyhexanoate (3HHx) by cultivation ofAeromonas hydrophila, fed-batch cultures were done under several nutrient limiting conditions. With the results from flask cultures, fed-batch cultures were carried out to produce large amounts of PHA. In the fed-batch culture, firstly glucose was fed to grow cell, and then, oleic acid fed to stimulate PHA in the cell. The final cell concentration, PHA content, PHA concentration, and 3-hydroxy-hexanoate fraction in 38 hr were 48.9 g/L, 15.05 wt%, 7.36 g/L and 12.2 wt%, respectively, resulting in the productivity of 0.19 g/L-h under phosphate-limiting condition.     

Enantioselective synthesis of ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE) from ethyl-3-oxo-3-phenylpropanoate using recombinant fatty acid synthase (FAS2) from Kluyveromyces lactis KCTC 7133 in Pichia pastoris GS115

Various yeast strains were examined for the microbial reduction of ethyl-3-oxo-3-phenylpropanoate (OPPE) to ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE), which is the chiral intermediate for the synthesis of a serotonin uptake inhibitor, Fluoxetine. Kluyveromyces lactis KCTC 7133 was found as the most efficient strain in terms of high yield (83% at 50 mM) and high optical purity ee > 99% of S-HPPE. Based on the protein purification, activity analysis and the genomic analysis, a fatty acid synthase (FAS) was identified as the responsible β-ketoreductase. To increase the productivity, a recombinant Pichia pastoris GS115 over-expressing FAS2 (α-subunit of FAS) of K. lactis KCTC7133 was constructed. In the optimized media condition, the recombinant P. pastoris functionally over-expressed the FAS2. Recombinant P. pastoris showed 2.3-fold higher reductase activity compared with wild type P. pastoris. With the recombinant P. pastoris, the 91% yield of S-HPPE was achieved at 50 mM OPPE maintaining the high optical purity of the product (ee > 99%).     

The accumulation of poly(3-hydroxyalkanoates) in Rhodobacter sphaeroides

In recent years industrial interest has been focussed on the evaluation of poly(3-hydroxyalkanoates) (PHA) as potentially biodegradable plastics for a wide range of technical applications. Studies have been carried out in order to optimize growth and culture conditions for the intracellular formation of PHA in the phototrophic, purple, non-sulfur bacterium Rhodobacter sphaeroides. Its potential to produce polyesters other than poly(3-hydroxybutyrate) (PHB) was investigated. On an industrial scale, the use of photosynthetic bacteria could harness sunlight as an energy source for the production of these materials. R. sphaeroides was grown anaerobically in the light on different carbon sources. Under nitrogenlimiting conditions a PHA content of up to 60 to 70% of the cellular dry weight was detected. In all of the cases studied, the storage polymer contained approximately 98 mol% of 3-hydroxybutyrate (HB) and 2 mol% 3-hydroxyvalerate (HV) monomer units. Decreasing light intensities did not stimulate PHA formation. Compared to Rhodospirillum rubrum (another member of the family of Rhodospirillaceae), R. sphaeroides showed a limited flexibility in its ability to form PHA with varying monomer unit compositions.